Its a 5 micron, sagittal brain section embedded in paraffin. I follow the protocol that i received with the Fluorojade B stain that I used. Its from Histochem Inc. I tried to increase the incubation period in 0.06% Potassium permanganate and 0.0004% FJB in 0.1% acetic acid to 30 mins. Most of the stain i find in the blood vessels and hence the neuron staining is much fainter.
1) How are you fixing your brains? Incomplete perfusion/fixation can lead to nonspecific staining in the vasculatrue, and 2) are you absolutely sure that you have cell death to be picked up by FluoroJade? I ask because in my experience when I have tissue with a lot of cell death then the neuronal staining is very clearly detectable far above background/vasculature labeling, however the vessels come out very bright in tissue where there's little-to-no cell death. Have you run this stain on a positive control? Sorry for asking more questions than giving answers, but it's all the advice I can think of!
Histochem protocol never works, i will try increasing FjB, decreasing permanganate to 0.03% or less and of course reducing time 10-15 min. It works in frozen sections
Kate Karelina, thank you so much for your insight. I will look into all these factors. Your questions actually answer most of my doubts.
- Badges
- Science topic
- Similar topics
- Correction